Isolation of whole virus and viral proteins is of most importance for the production of vaccines useful as prophylactic or therapeutic vaccines against viral infections. Typically, the immune response elicited by virus vaccines is based on raising an appropriate cellular and humoral immunity against immunogenes of the virus. For example, for influenza virus, the envelope proteins or surface proteins neuraminidase (NA), hemagglutinin (HA) and the ion channel (M2) are regarded as the most prominent candidate vaccines. In case of e.g. HIV gp120 and gep41 are assumed to represent vaccine candidates.
Influenza infection is a wide spread contagious disease of the respiratory tract. Due to annual death rates and its potential to cause pandemics, influenza remains a major public health concern. To control influenza outbreaks prophylactic vaccinations in conjunction with anti-viral medications are regarded as being most promising.
The same is true for vaccinia virus infection. To control or eradicate new outbreaks of vaccinia virus and other small pox virus, prophylactic vaccinations are required.
Influenza and small pox are two severe infectious diseases that can lead to death. Vaccine doses are currently produced for both diseases. While several million humans are getting annually vaccinated against influenza, the vaccinia virus vaccines are produced to be stocked in case of an emerging pandemic.
Today efforts are undertaken to switch from conventional vaccine production processes, e.g. virus production in primary cell lines (for vaccinia virus) or embryonated hen's egg (for influenza virus), to continuous cell line cultures. However, these require new downstream processing strategies allowing isolation and purification of the whole virus or virus components, in particular, of viral membrane proteins which are the main immunogens of the virus. Various approaches have been suggested for purification of whole virus and virus proteins. For example, WO 96/15231 provides a general overview of methods useful for producing biologicals in protein free cultures. Said methods comprise pseudo-affinity purification steps as well as other chromatography methods, enzyme treatment steps, ultra-filtration step and hydrophobic interaction steps.
In EP 171 086 a method for purification of influenza virus is disclosed. Said method comprises subjecting a solution containing the influenza virus to column chromatography using, as a gel for a chromatography, a sulfuric acid ester of cellulose or a cross-linked polysaccharide.
In said document, the modification and functionalization of a chromatography gel useful for pseudo-affinity column chromatography for influenza virus is described. The gel is a sulfuric acid ester of cellulose or a cross-linked polysaccharide. The steps for functionalization of the cellulose comprises reacting pyridine with chloro-sulfonic acid at a temperature below 0° C., thereafter heating said mixture to a temperature of about 65 to 70° C. and maintaining said temperature while adding the crystalline cellulose gel and stirring the mixture for another three hours at a temperature of about 65 to 70° C. Thereafter, the mixture is cooled, neutralized and a gel is obtained useful for column chromatography.
However, major drawbacks of applying column chromatography with cellulose gel include long process time with large recovery liquid volume. In addition, the flow rates of the column chromatography are limited. Further, the preparation of the columns is time consuming and cumbersome and requires regeneration steps. Thus, the state of the art method of an unit operation often used in influenza-purification comprising column chromatography based on the sulfated cellulose gel, known as Cellufine™ sulfate has major disadvantages due to the limited flow rate in view of the high backpressure, thus, leading to sub-optimal productivity rates of the process.
It is therefore an object of the present invention to provide a method for purification of whole virus or virus proteins in a simple manner overcoming the drawbacks in the art.
It is another object of the present invention to provide material useful for said purification, namely to provide material useful for pseudo-affinity purification of virus and viral proteins.